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Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation

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Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation International Journal of Molecular Sciences Review Structural Perspectives on the Mechanism of Soluble Guanylate Cyclase Activation Elizabeth C. Wittenborn and Michael A. Marletta * California Institute for Quantitative Biosciences, Departments of Chemistry and of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; [email protected] * Correspondence: [email protected] Abstract: The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo–electron microscopy (cryo-EM) enabled visualization of full-length sGC for the first time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation. Keywords: nitric oxide; soluble guanylate cyclase; cryo–electron microscopy; enzyme structure Citation: Wittenborn, E.C.; Marletta, 1. Introduction M.A. Structural Perspectives on the Soluble guanylate cyclase (sGC) is a nitric oxide (NO)-responsive enzyme that serves Mechanism of Soluble Guanylate as a source of the secondary messenger cyclic guanosine monophosphate (cGMP) in Cyclase Activation. Int. J. Mol. Sci. humans and other higher eukaryotes [1]. Upon NO binding to sGC, the rate of cGMP 2021, 22, 5439. https://doi.org/ formation increases by several hundred-fold, effectively amplifying the initial NO signal. 10.3390/ijms22115439 cGMP in turn regulates downstream effectors, such as cGMP-dependent protein kinases (PKGs), ion channels, and phosphodiesterases (PDEs), leading to a variety of physiological Academic Editor: Jens Schlossmann responses that include vasodilation, inhibition of platelet aggregation, and effects on neu- rotransmission [2,3]. As a result, dysregulation of the NO-sGC-cGMP signaling pathway Received: 3 May 2021 is associated with various disease pathologies, including hypertension, cardiovascular Accepted: 18 May 2021 Published: 21 May 2021 diseases, neurodegenerative diseases, and asthma [4–8]. Indeed, stimulators of sGC activity have proven effective in the treatment of pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. The first sGC-targeted drug, Adempas®, was Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in approved by the US Food and Drug Administration in 2013 [9]. Given this clinical relevance, published maps and institutional affil- sGC has received considerable attention over the years, with research efforts aimed at better iations. understanding the complex mechanism of activity regulation. Biochemical aspects of sGC activation and deactivation have been reviewed recently [10] and this review will focus on recent and exciting advances in our knowledge of sGC from a structural perspective. 2. Domain Architecture of sGC Copyright: © 2021 by the authors. α β Licensee MDPI, Basel, Switzerland. sGC is an obligate heterodimer consisting of homologous and subunits that share This article is an open access article roughly 30% sequence identity, depending on the species of origin. Each subunit contains distributed under the terms and four domains: an N-terminal heme NO/O2-binding (H-NOX) domain, followed by a conditions of the Creative Commons Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal catalytic (CAT) Attribution (CC BY) license (https:// domain (Figure1A). As its name implies, the H-NOX domain contains a heme cofactor creativecommons.org/licenses/by/ that binds a single equivalent of NO at the iron center [11–13]. Although both α and β 4.0/). subunits contain an H-NOX domain, only the β H-NOX domain contains a heme [12,14]. Int. J. Mol. Sci. 2021, 22, 5439. https://doi.org/10.3390/ijms22115439 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18 Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal catalytic (CAT) do- Int. J. Mol. Sci. 2021, 22, 5439 main (Figure 1A). As its name implies, the H-NOX domain contains a heme cofactor2 ofthat 18 binds a single equivalent of NO at the iron center [11–13]. Although both α and β subunits contain an H-NOX domain, only the β H-NOX domain contains a heme [12,14]. PAS do- PASmains domains are widely are widely found foundin signaling in signaling proteins proteins where where they serve they serveas sensing as sensing domains domains or as ormediators as mediators of protein–protein of protein–protein interactions interactions [15]. CC [15 domains]. CC domains are versatile are versatile structural structural motifs motifsthat vary that greatly vary greatlyin both in size both and size function, and function, but arebut commonly are commonly found C-terminal found C-terminal to PAS todomains PAS domains where wherethey are they thought are thought to contribute to contribute to signal to signal transduction transduction between between the PAS the PASdomain domain and andan effector an effector domain domain [15–17]. [15–17 Th]. Thee sGC sGC CAT CAT domain domain is is a a class class III nucleotidenucleotide cyclasecyclase domaindomain and and is is the the site site of of cGMP cGMP formation formation from from guanosine guanosine triphosphate triphosphate (GTP) (GTP) [18]. A[18]. single A single active active site issite formed is formed at the at theα/ βα/dimerβ dimer interface, interface, with with critical critical catalytic catalytic residuesresidues contributedcontributed by eacheach subunitsubunit [[19–22].19–22]. AA second,second, catalyticallycatalytically inactiveinactive nucleotidenucleotide bindingbinding site,site, termed the pseudosymmetric pseudosymmetric site, site, is is also also present present at at the the CAT CAT domain domain interface interface and and is isproposed proposed to be to involved be involved in allosteric in allosteric activity activity regulation regulation by adenosine by adenosine triphosphate triphosphate (ATP) (ATP)[23–25]. [23 –25]. Figure 1.1. An introduction toto sGC.sGC. ((AA)) sGCsGC domaindomain architecture.architecture. sGCsGC isis aa heterodimerheterodimer composedcomposed ofof homologous α and β subunits. Each subunit contains four domains: an N-terminal heme NO/O2- homologous α and β subunits. Each subunit contains four domains: an N-terminal heme NO/O2- binding (H-NOX) domain, a Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-termi- binding (H-NOX) domain, a Per-Arnt-Sim (PAS) domain, a coiled coil (CC) domain, and a C-terminal nal catalytic (CAT) domain. The red trapezoid represents the heme cofactor found in the β H-NOX catalytic (CAT) domain. The red trapezoid represents the heme cofactor found in the β H-NOX domain. Residue numbering of the domains corresponds to the sequence of human sGC. (B) Typical domain.activity profile Residue of numbering sGC. sGC ofexhi thebits domains a low, corresponds basal level toof theactivi sequencety in the of humanabsence sGC. of NO (B) Typical(U: un- activityliganded). profile Addition of sGC. ofsGC a single exhibits equivalent a low, basal of NO level (1-NO) of activity results in in the a modest absence increase of NO (U: in unliganded).activity. Full AdditionsGC activity of a is single obtained equivalent in the ofpr NOesence (1-NO) of a resultsmolar inexcess a modest of NO increase (xsNO). in Activity activity. Fullcan also sGC be activity stim- isulated obtained by the in small the presence molecule of 3-(5 a molar′-hydroxymethyl-2 excess of NO′ (xsNO).-furyl)-1-benzylindazole Activity can also (YC-1). be stimulated (C) UV-Visible by the smallabsorption molecule spectra 3-(5 of0-hydroxymethyl-2 ligand binding to0 -furyl)-1-benzylindazolethe sGC heme. Upon NO (YC-1).binding, ( Cthe) UV-Visible Soret peak absorptionshifts from spectra~430 to of399 ligand nm. The binding inset toshows the sGCthe Q-band heme. Uponregion NOof the binding, spectra. the Figure Soret adapted peak shifts from from reference ~430 to[11] 399 [Creative nm. The Commons inset shows Attribution the Q-band license] region. of the spectra. Figure adapted from reference [11] [Creative Commons Attribution license]. 3. Activity Profile of sGC 3. ActivityIn vitro, Profile sGC exhibits of sGC a low basal level of activity in the absence of any ligand (Figure 1B) [23,26,27].In vitro, sGC An increase exhibits in a sGC low activity basal level is observed of activity upon in the the addition absence of of NO, any either ligand in (Figurethe form1B) of [NO23, 26gas,27 or]. through An increase the use in of sGC small activity molecule is observed NO donors, upon such the as addition NONOates of NO,[23,26–28]. either Interestingly, in the form ofthe NO extent gas of or enzyme through activation the use ofdepends small on molecule the relative NO donors,amount suchof NO as added. NONOates A modest [23,26 (~4-fold)–28]. Interestingly, increase in sG theC activity extent ofis observed enzyme activationin the presence depends of a onsingle the molar relative equivalent amount ofof NONO, added. which Ais modesttermed the (~4-fold) 1-NO increasestate (Figure in sGC 1B) activity [23,26,27]. is ob- In servedthis state, in theNO presenceforms a 5-coordinate of a single molarFe2+–NO equivalent complex ofwith NO, the which heme iscofactor termed of the the 1-NO β H- state (Figure1B) [23,26,27]. In this state, NO forms a 5-coordinate Fe 2+–NO complex with the heme cofactor of the β H-NOX domain, which is discernable by ultraviolet-visible (UV-Vis) absorption spectroscopy as a shift in the Soret band of the heme from ~430 nm 2+ (for the Fe unliganded species; the exact λmax depends on the species of sGC) to 399 nm (Figure1C) [ 29,30].
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